Departments of Developmental Biology and Genetics, Stanford University School of Medicine, Stanford, California 94305 annev@stanford.edu.

Abstract

Meiotic recombination is initiated by the programmed induction of double-strand DNA breaks (DSBs), lesions that pose a potential threat to the genome. A subset of the DSBs induced during meiotic prophase become designated to be repaired by a pathway that specifically yields interhomolog crossovers (COs), which mature into chiasmata that temporarily connect the homologs to ensure their proper segregation at meiosis I. The remaining DSBs must be repaired by other mechanisms to restore genomic integrity prior to the meiotic divisions. Here we show that HIM-6, the Caenorhabditis elegans ortholog of the RecQ family DNA helicase BLM, functions in both of these processes. We show that him-6 mutants are competent to load the MutSγ complex at multiple potential CO sites, to generate intermediates that fulfill the requirements of monitoring mechanisms that enable meiotic progression, and to accomplish and robustly regulate CO designation. However, recombination events at a subset of CO-designated sites fail to mature into COs and chiasmata, indicating a pro-CO role for HIM-6/BLM that manifests itself late in the CO pathway. Moreover, we find that in addition to promoting COs, HIM-6 plays a role in eliminating and/or preventing the formation of persistent MutSγ-independent associations between homologous chromosomes. We propose that HIM-6/BLM enforces biased outcomes of recombination events to ensure that both (a) CO-designated recombination intermediates are reliably resolved as COs and (b) other recombination intermediates reliably mature into noncrossovers in a timely manner.

The him-6 mutant is proficient for timely loading of RAD-51 and MSH-5 at nascent recombination sites. Immunofluorescence images showing RAD-51 and MSH-5 foci in a him-6 mutant gonad, revealing dynamics of RAD-51 and MSH-5 foci that are similar to wild type (shown in Figure S1). Images show a region of the gonad extending from meiotic prophase entry (left) until the end of the pachytene stage (right). RAD-51 foci are abundant during early pachytene, decline during mid-pachytene, and are nearly absent by late pachytene; faint MSH-5 foci appear during early pachytene, become brighter and more abundant during mid-pachytene, and then reduce in number at late pachytene, where they persist at crossover-designated sites. At right, insets of the indicated fields show that although both RAD-51 and MSH-5 foci are present in the same nuclei, they rarely overlap. Scale bar, 10 μm.

The him-6 mutant is proficient for crossover designation and crossover regulation. (A) Immunofluorescence images of late pachytene nuclei from a him-6 mutant gonad, showing that each nucleus has six GFP::COSA-1 foci, each associated with a comet-like ZHP-3 signal; the him-6 mutant is cytologically indistinguishable from wild type, where GFP::COSA-1 foci reflect designation of a single cytologically differentiated CO site on each homolog pair (). (B) Graph showing quantitation of GFP::COSA-1 foci in late pachytene nuclei from wild-type (n = 81) and him-6 (n = 94) worms; error bars indicate standard deviation. (C) GFP::COSA-1 and ZHP-3 immunofluorescence in late pachytene nuclei from a him-18 mutant, which is defective at a late step in crossover formation. Scale bar, 10 μm.

Timely transition in pachytene progression in the him-6 mutant. (A) Immunoflouorescence images of whole-mount gonads, extending from the distal premeiotic tip to the end of the pachytene region. In both the wild-type and him-6 mutant, phosphorylation of nuclear envelope protein SUN-1 (SUN-1 S8Pi) and association of the DSB-promoting protein DSB-2 with chromatin show similar dynamics: They are detected in germ-cell nuclei at the onset of meiotic prophase and then decline and disappear from most nuclei during mid-pachytene. As SUN-1 S8Pi and DSB-2 persist until late pachytene in multiple mutants that fail to make crossover-eligible recombination intermediates (; ) (see B), this finding is consistent with the conclusion that him-6 mutants are proficient for generating crossover intermediates. Scale bar, 10 μm. (B) Bar graph showing quantitation of the percentage of the meiotic zone occupied by SUN-1 S8-Pi-positive nuclei in germ lines of indicated genotypes. The presence/absence of SUN-1 S8-Pi signals was assessed in the portion of the germ line extending from the onset of meiotic prophase to the end of the pachytene region. The extent of the SUN-1 S8-Pi-positive zone was defined as the number of contiguous rows of nuclei in which all rows contained two or more nuclei with SUN-1 S8-Pi staining/total rows of nuclei in the scored region. Data are represented as mean ±SD. Whereas the SUN-1 S8-Pi-positive zones were significantly extended in the cosa-1 and cosa-1; him-6 mutants relative to wild type and the him-6 single mutant (P < 0.0001, two-tailed Mann–Whitney tests), no significant difference was observed between wild type and the him-6 mutant (P = 0.10). Numbers of germ lines scored: wild type, 18; him-6, 21; cosa-1, 20; cosa-1; him-6, 17.

Dissociation of some chromosome pairs into univalents by the end of diakinesis in him-6 mutants. (A) Diplotene and diakinesis-stage oocytes from the him-6 mutant, stained with antibodies against chromosome axis proteins HTP-1/2 and SC central region protein SYP-1. The chromosomes appear indistinguishable from wild type at the diplotene stage (), with the SYP-1 and HTP-1/2 proteins localizing to reciprocal domains as the chromosomes desynapse. By diakinesis, a mixture of bivalents and univalents (arrowheads) are detected; moreover, the univalents exhibit a reciprocal localization pattern for HTP-1/2 and SYP-1 that is normally associated with a crossover/chiasma. Right: For the top two diakinesis nuclei, the insets show selected univalents. (B) Full chromosome complements of individual him-6 diakinesis oocytes (from a single germ line, shown below). Oocyte nuclei from left to right were in the −4, −3, −2, and −1 positions relative to the spermatheca, with the −1 oocyte being the most mature. Arrows indicate univalents that have ZHP-3 foci, which normally mark crossover sites. Scale bars, 5μm. (C) Graph showing that the incidence of univalents in the him-6 mutant increases as oocytes progress through the diakinesis stage. The him-17(e2707) mutant, in which a decrease in DSB formation is responsible for the reduction in crossovers/chiasmata, is used as a control; in the him-17(e2707) control, there is a modest increase in the number of univalents scored during progression from the −3 to −1 position, reflecting improved ability to detect univalents as chromosome compaction increases during oocyte maturation. In contrast, there is a larger and more significant increase in univalents observed between the −3 and −1 oocytes in the him-6 mutant, suggesting the presence of more persistent connections that eventually dissociate (only a subset of P-values is depicted; see text). Error bars indicate SEM. Numbers of nuclei scored: him-17, n = 95; him-6, n = 83.

Occurrence of achiasmate X chromosomes in him-6 mutant oocytes correlates with reduced crossover frequency during oocyte meiosis. (A) Each column shows the full karyotype of a single diakinesis oocyte from a him-6 mutant worm, with the X chromosomes visualized by chromosome paint. In the top two oocytes, the X chromosome pairs comprise a bivalent, whereas in the bottom oocyte, the X chromosomes clearly lack a chiasma and are present as univalents. (The different configurations of the red and green paint signals on the X chromosome bivalents in the top two images likely reflect different CO positions.) Scale bars, 5 μm. (B) Graph showing the genetic map distances measured for oocyte meiosis for the dpy-3 unc-3 interval on the X chromosome; error bars indicate 95% confidence intervals. Although the reduction in CO frequency in the tested interval in the him-6 mutant appears nominally higher than the incidence of achiasmate X chromosomes observed using the paint assay, based on the experimental error associated with both types of measurements, there is insufficient statistical power to conclude that this difference is significant. Thus, these data are not considered as part of the evidence for persistent connections between noncrossover chromosomes.